Hevein-binding monoclonal antibodies

ABSTRACT

This invention relates to antibody engineering technology. More particularly, the present invention relates to human IgE antibodies and derivatives thereof, which bind allergenic hevein with high affinity and specificity. The present invention also relates to processes for makings and engineering such hevein-binding monoclonal antibodies and to methods for using these antibodies and derivatives thereof in the field of immunodiagnostics, enabling qualitative and quantitative determination of allergenic hevein in biological and raw material samples, as well as in immunotherapy, enabling blocking of allergenic hevein in allergic patients.

FIELD OF THE INVENTION

This invention relates to antibody engineering technology. Moreparticularly, the present invention relates to human IgE antibodies andderivatives thereof, which bind allergenic hevein with high affinity andspecificity. The present invention also relates to processes for makingand engineering such hevein-binding monoclonal antibodies and to methodsfor using these antibodies and derivatives thereof in the field ofimmunodiagnostics, enabling qualitative and quantitative determinationof allergenic hevein in biological and raw material samples, as well asin immunotherapy, enabling blocking of allergenic hevein in allergicpatients.

BACKGROUND OF THE INVENTION

Almost 20% of the population world-wide are suffering from allergy.Consequently, it is a health problem of increasing seriousness. Allergyis a hypersensitivity reaction against substances in air, food or water,which are normally harmless (Corry and Kheradmand, 1999). A new andforeign external agent triggers an allergic reaction, which aims atdisposal of that agent from the body. In IgE-mediated allergicreactions, also called immediate or type I hypersensitivity reactions,under the first exposure of a foreign substance, allergen, to the body,IgE-bearing B-cells begin to produce soluble IgE molecules which willthen bind to high-affinity IgE receptors present on the surface of awide variety of cells, most importantly to mast cells. If the sameforeign substance is encountered again, the cross-linking of thereceptor-bound IgE molecules by the allergen occurs, resulting incellular activation followed by the release of toxic products such ashistamines, which will elicit the signs and symptoms of an allergicreaction.

Latex allergy is a serious medical problem with an increasing number ofpatients (Slater, 1994, Turjanmaa et al., 1996). Latex is a complexintracellular product, a milky sap, produced by the laticiferous cellsof the rubber tree, Hevea brasiliensis, which is used in a variety ofeveryday articles, e.g. for the production of gloves, balloons, andcondoms, and in manufacturing of medical devices. Latex allergy is aserious problem especially with health-care workers, rubber industryworkers and patients having undergone several surgical procedures. Latexallergy has also been reported to be associated with pollen allergiesand food allergies (Nel and Gujuluva, 1998). The cross-reactivitybetween latex and food allergens is established as the latex-fruitsyndrome that might be the consequence of hevein-like protein domains orsimilar epitopes (Brehler et al., 1997, Chen et al., 1998, Mikkola etal., 1998). Many latex proteins have been identified as allergens(Breiteneder and Scheiner, 1998). One of the major latex allergens ishevein, which is a defence protein involved in, for instance, theinhibition of several chitin-containing fungi (Lee et al., 1991, Aleniuset al., 1996, Chen et al., 1997). Hevein is a small chitin-bindingprotein of 43 amino acids with four disulphide bonds. Itsthree-dimensional structure has been determined by X-ray diffraction andNMR (Rodriguez-Romero et al., 1991; Andersen et al., 1993).

IgE antibodies distinctively recognise allergenic epitopes, which wouldbe useful in clinics or immunodiagnostics for detecting and determiningallergen concentrations of complex materials. Further, allergenicepitopes are usually different from the immunogenic epitopes ofproteins. This fact has hampered the production of monoclonal antibodiescapable of specific binding of allergenic epitopes by conventionalmethodology such as hybridoma technology. It has been recently shownthat the development of allergen-specific IgE antibodies is possible bythe phage display technology (Steinberger et al., 1996). Thismethodology is giving new tools to produce allergen-specific recombinantantibodies that can be produced in consistent quality for clinical anddiagnostic applications.

SUMMARY OF THE INVENTION

We describe in this application the development and characterisation ofhuman IgE antibody fragments that bind allergenic hevein with affinityand specificity high enough to be utilised as reagents in immunoassaysdesigned for the qualitative and quantitative measurement of hevein inbiological samples and, in immunotherapy of allergic patients.Specifically, the present invention describes selection of human IgEantibodies specific to hevein by the phage display technique, and thecharacterisation of the binding properties of the engineered antibodyfragments produced in E.coli.

This invention thus provides new reagents to be utilised in differentkinds of immunoassay protocols, as well as human immunotherapy. Theinvention also permits guaranteed continuous supply of these specificreagents of uniform quality, eliminating inherent batch-to-batchvariation of polyclonal antisera. These advantageous effects permit themanufacture of new, specific and economical immunodiagnostic assays ofuniform quality.

Consequently, one specific object of the present invention is to providehuman IgE mono-clonal antibodies, fragments thereof, orother-derivatives of such antibodies, which bind hevein with affinityand specificity high enough to allow qualitative and quantitativemeasurement of hevein in biological samples, as well as their use inimmunotherapy. The monovalent antibodies of the present inventiondemonstrate a specific binding to allergenic hevein.

Another object of the present invention is to provide cDNA clonesencoding hevein-specific antibody chains, as well as constructs andmethods for expression of such clones to produce hevein-bindingantibodies, fragments thereof or other derivatives of such antibodies.

A further object of this invention is to provide methods of using suchhevein-binding antibodies, fragments thereof or other derivatives ofsuch antibodies, or combinations of them for qualitative andquantitative measurement of hevein in biological samples. Additionally,this invention provides hevein-binding antibodies, fragments thereof orother derivatives of such antibodies, or combinations of them forimmunotherapy in allergic patients.

Other objects, features and advantages of the present invention will bebecome apparent from the following drawings and detailed description. Itshould be understood, however, that the detailed description and thespecific examples, while indicating preferred embodiments of theinvention, are given for illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures of the constructions are not in scale.

FIG. 1 shows a schematic presentation of an intact human IgE subclassantibody, Fab fragment and single-chain antibody (scFv). Theantigen-binding site is indicated by a triangle.

FIG. 2 shows schematically the panning procedure.

FIG. 3 shows a schematic presentation of the scFv phage display vectorused for the construction of scFv phage libraries.

FIG. 4 shows the deduced amino acid sequence of the heavy chain variableregion of the 1A4 and 1C2 antibodies. The Complementarity DeterminingRegions (CDRs) are underlined. Numbering is according to Kabat (Kabat etal., 1991).

FIG. 5 shows the deduced amino acid sequence of the light chain variableregion of the 1A4 and 1C2 antibodies. CDRs are underlined. Numbering isaccording to Kabat (Kabat et al., 1991).

FIG. 6 a shows the curve obtained from the competitive ELISA of 1A4 Fabfragment with human IgG1 subtype whose binding to hevein has beeninhibited by latex polypeptide.

FIG. 6 b shows the curve obtained from the competitive ELISA of 1 C2 Fabfragment with human IgG1 subtype whose binding to hevein has beeninhibited by latex polypeptide.

FIG. 7 shows the result of the competitive ELISA. The binding of 1A4 Fabfragments with human IgG1 subtype to hevein is inhibited by allergenicepitopes (6-mer and 13-mer) of the hevein.

ABBREVIATIONS

-   -   cDNA complementary deoxyribonucleic acid    -   CDR complementarity determining region    -   DNA deoxyribonucleic acid    -   E. coli Escherichia coli    -   ELISA enzyme-linked immunosorbent assay    -   Fab fragment with specific antigen binding    -   Fd variable and first constant domain of a heavy chain    -   Fv variable regions of an antibody with specific antigen binding    -   GFP green fluorescent protein    -   IgE immunoglobulin E    -   mRNA messenger ribonucleic acid    -   NMR nuclear magnetic resonance    -   PCR polymerase chain reaction    -   RNA ribonucleic acid    -   scfv single-chain antibody    -   supE⁻ a genotype of bacterial strain carrying a        glutamine-inserting amber suppressor tRNA    -   V_(H) variable region of a heavy chain    -   V_(L) variable region of a light chain

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are provided for some terms used in thisspecification. The terms, “immunoglobulin”, “heavy chain”, “light chain”and “Fab” are used in the same way as in the European Patent ApplicationNo. 0125023.

“Antibody” in its various grammatical forms is used herein as acollective noun that refers to a population of immunoglobulin moleculesand/or immunologically active portions of immunoglobulin molecules,i.e., molecules that contain an antigen binding site or a paratope.

An “antigen-binding site”, a “paratope”, is the structural portion of anantibody molecule that specifically binds an antigen.

Exemplary antibodies are those portions of an immunoglobulin moleculethat contain the paratope, including those portions known as Fab and Fv.

“Fab” (fragment with specific antigen binding), a portion of antibodiescan be prepared by the proteolytic reaction of papain on substantiallyintact antibodies by methods that are well known. See for example, U.S.Pat. No. 4,342,566. Fab fragments can also be produced by recombinantmethods, which are well known to those skilled in the art. See, forexample, U.S. Pat. No. 4,949,778.

“Domain” is used to describe an independently folding part of a protein.General structural definitions for domain borders in natural proteinsare given in Argos, 1988.

A “variable domain” or “Fv” is used to describe those regions of theimmunoglobulin molecule, which are responsible for antigen or haptenbinding. Usually these consist of approximately the first 100 aminoacids of the N-termini of the light and the heavy chain of theimmunoglobulin molecule.

“Single-chain antibody” (scFv) is used to define a molecule in which thevariable domains of the heavy and light chain of an antibody are joinedtogether via a linker peptide to form a continuous amino acid chainsynthesised from a single mRNA molecule (transcript).

“Linker” or “linker peptide” is used to describe an amino acid sequencethat extends between adjacent domains in a natural or engineeredprotein.

A “hevein-binding antibody” is an antibody, which specificallyrecognises hevein and binds to it, due to interaction mediated by itsvariable domains.

As examples of fragments of such antibodies falling within the scope ofthe invention we disclose here scFv fragments of 1A4 and 1C2 as shown inFIGS. 4 and 5. In one preferred embodiment, the present invention thusprovides derivatives of hevein-binding antibodies, e.g. Fab fragments orscFv fragments. It will be appreciated that mutant versions of the CDRsequences or complete V_(L) and V_(H) sequences having one or moreconservative substitutions which do not substantially affect bindingcapability, may alternatively be employed.

For use in immunoassay, e.g. for qualitative or quantitativedetermination of hevein in biological samples, antibodies and antibodyderivatives of the invention may be labelled. For these purposes, anytype of label conventionally employed for antibody labelling isacceptable.

For use in immunotherapy, e.g. for blocking allergenic hevein inallergic patients, antibodies and antibody derivatives of the inventionmay be labelled. For these purposes, any pharmaceutically acceptablelabel conventionally employed for antibody labelling is appropriate.

In another aspect, the present invention also provides DNA moleculesencoding an antibody or antibody derivative of the invention, andfragments of such DNAs, which encode the CDRs of the V_(L) and/or V_(H)region. Such a DNA may be cloned in a vector, more particularly, forexample, an expression vector which is capable of directing expressionof antibody derivatives of the invention, or at least one antibody chainor a part of one anti-body chain.

In a further aspect of the invention, host cells are provided, selectedfrom bacterial cells, yeast cells, fungal cells, insect cells, plantcells and mammalian cells, containing a DNA molecule of the invention,including host cells capable of expressing an antibody or antibodyderivative of the invention. Thus, antibody derivatives of the inventionmay be prepared by culturing host cells of the invention expressing therequired antibody chain(s), and either directly recovering the desiredprotein or, if necessary, initially recovering and combining individualchains.

The above-indicated scFv fragments were obtained by biopanning of ahuman IgE scFv-phage library using allergenic recombinant hevein. Thehuman IgE scFv-phage library was constructed from mRNAs isolated fromlymphocytes of a latex-allergic patient. The variable region of thelight and heavy chain cDNAs were synthesised using human IgE-specificprimers for Fd cDNAs and human kappa (κ) and lambda (λ) light chainsusing human κ and λ chain specific primers. The variable regions of thelight and heavy chains were amplified by PCR using human κ and λ chainspecific primers for Vκ and Vλ cDNAs and human IgE specific primers forV_(H) cDNAs, respectively. The human IgE scFv library was constructed bycloning the variable region cDNAs into a scFv phage display vector usingrestriction sites introduced into the PCR primers.

The human IgE scFv library was selected by phage display using a panningprocedure. The human IgE scFv phage library was screened by abiotinylated allergenic recombinant hevein in solution and the binderswere captured on streptavidin. The elution of phages was done with 100mM HCl .(PH 2.2) followed by immediate neutralisation with 2 M Trissolution. The phage eluate was amplified in E. coli cells. After 5rounds of biospanning, soluble scFv fragments were produced fromisolated phages. The binding specificity of the selected scFv fragmentswas analysed by ELISA. Several hevein-specific scFv fragment clones wereobtained.

As described herein, the phage display technique is an efficient andfeasible approach to develop human IgE recombinant anti-heveinantibodies for diagnostic and therapeutic applications.

While one successful selection strategy for obtaining antibody fragmentsof the invention has been described, numerous variations, by whichantibody fragments of the invention may be obtained, will be apparent tothose skilled in the art. It may prove possible to select scFv fragmentsof the invention directly from a phage or microbial display library ofscFv fragment or its derivatives. A phage or microbial cell, whichpresents a scFv fragment or other antibody fragment of the invention asa fusion protein with a surface protein, represents a still furtheraspect of the invention.

While microbial expression of antibodies and antibody derivatives of theinvention offers means for efficient and economical production of highlyspecific reagents of uniform quality suitable for use inimmunodiagnostic assays and immunotherapy, alternatively it may provepossible to produce such a reagent, or at least a portion thereof,synthetically. By applying conventional genetic engineering techniques,initially obtained antibody fragments of the invention may be altered,e.g. new sequences linked, without substantially altering the bindingcharacteristics. Such techniques may be employed to produce novelhevein-binding hybrid proteins, which retain both affinity andspecificity for hevein as defined hereinbefore.

The development and characterisation of the human hevein-bindingrecombinant antibodies and their usefulness in immunoassays is nowdescribed in more detail in the following examples.

EXAMPLE 1 The Recombinant Hevein-Specific scfv Fragment by Phage DisplaySelection

In this example the human IgE scFv library was constructed and selectedby allergenic hevein in order to isolate scFv fragments with affinityand specificity to hevein. Construction of human IgE scFv phage librarywas prepared indirectly by constructing IgE Fab-κ and Fab-λ librariesfirst, and then the particular library DNAs were used for PCRamplification of variable domains of heavy and light chains.

I. Construction of the Human IgE scFv Phage Libraries

100 ml of heparinised blood was obtained from a latex-allergic patient.Lymphocytes were isolated according to an Ig-Prime kit protocol(Novagen). Per 10 ml of blood 30 ml of lysis buffer (155 mM NH₄Cl, 10 mMNH₄CO₃, 0.1 mM EDTA, pH 7.4) was added and incubated on ice for 15 minwith shaking occasionally. After centrifugation at 450 g for 10 mm thelymphocytes, i.e. the white blood cell pellet, were collected. Thepellet was washed twice with lysis buffer and after the finalcentrifugation the lymphocyte pellet was resuspended in D-solution.Lymphocyte RNAs were isolated using Promega's RNAgents Total RNAIsolation kit according to the manufacturer's protocol. The first strandcDNA synthesis was carried out using Promega's Reverse Transcriptionsystem kit. For the synthesis of Fd-fragment cDNA and light chain cDNAsthe primers of the constant region of the epsilon (ε) chain (Cε1 andCε2) and the primer of the kappa (Cκ1) and lambda (Cλ1) chain were used,respectively. Primers used for the cDNA synthesis and PCR amplificationsof human IgE Fd region and light chains are showed in Table I and TableII.

PCR amplifications were carried out in two steps: a primary PCR foramplifying Fd and light chains from cDNA templates and a secondary PCRfor adding restriction sites to the 5′-end of the DNA fragments obtainedafter a primary PCR. First the Fd region was amplified by PCR using theprimers specific for the variable region of the heavy chains (VH1a-VH7a)and Cε1NotI primer. Accordingly, the kappa and lambda light chains wereamplified using specific primers for variable region of the light chains(Vε1a-Vκ6b and Vλ1a-Vλ10) and Cε1NotI primer, respectively. Primers forthe secondary PCR were Cκ1 and Vκ/λ1 and Cε2 for the Fd region, Vκ/λ1and Cλ1 for the kappa light chain and Vλ1A and Cκ/λ1 for the lambdalight chain. The primary PCR amplification was done at the followingconditions: 1 cycle of 3 min at 93° C. for denaturation, 7 cycles of 1min at 93° C., 30 s at 63° C. and 50 s at 58° C. for annealing and 1 minat 72° C. for elongation, 23 cycles of 1 min at 93° C., 30 s at 63° C.and 1 min at 72° C. followed by 1 cycle of 10 min at 72° C. For thesecondary PCR the amplification conditions were as follows: 1 cycle of 3min at 95° C. for denaturation, 25 cycles of 1.5 min at 94° C., 1 min at65° C. for annealing and 1.5 min at 72° C. for elongation followed by 1cycle of 10 min at 72° C. Between the primary and the secondary PCR andafter the secondary PCR tie amplified DNA fragments were purified.

The final PCR products of the different antibody fragments were pooledand digested with appropriate restriction enzymes. Digested DNAfragments, encoding IgE Fd region and κ and λ light chains, were ligatedinto a phagemid vector and transformed into E. coli XL-1 Blue cells toyield an Fab-κ and Fab-λ libraries of 10⁶ independent clones. To avoidpossible problems on the expression of Fab fragments on a phage particlean antibody library in scFv format was constructed. Phagemid DNAs fromdifferent libraries were isolated and used as template DNAs foramplifying the variable regions of the human IgE heavy and human lightchains in order to construct human IgE scFv-κ and scFv-λ libraries.

PCR amplification of the variable region of the heavy chain was carriedout using human V_(H) specific primers (VH1-VH4 and VH1A). Amplificationof the variable region of the light chains was done using the followingprimer pairs: Vκ1-Vκ7, Vκ2-Vκ8, Vκ3-Vκ9, Vκ4-Vκ10, Vκ5-Vκ11 and Vκ6-Vκ11for human kappa chain and Vλ1-Vλ8, Vλ2-Vλ9, Vλ3-Vλ9, Vλ4-Vλ9, Vλ5-Vλ10,Vλ6-Vλ10 and Vλ7-Vλ10 for human lambda chain (see Tables III and IV).The amplified DNA fragments were purified and digested in order toligate into a scFv phage display vector (FIG. 3). Ligation mixtures weretransformed into E. coli XL-1 Blue cells resulting in the human IgEscFv-κ and scFv-λ libraries with approximately 10⁵ independent clones.

II. Selection of the Human scFv-Libraries

The human scFv-κ and scFv-λ libraries were selected by the phage displaytechnique (McCafferty et al., 1990, Barbas et al., 1991). To isolatehevein-binding antibody fragments, the human IgE scFv-κ and scFv-λlibraries displayed on the surface of the bacteriophage were pooled andpanned using an affinity panning procedure (FIG. 2). First the phagepools were allowed to react either with biotinylated, immunoreactivehevein or with a biotinylated control protein (background) for 1.5 h.Thereafter, the phage pools were transferred to microtitre plate wellscoated with biotin binding streptavidin. After a 30-min incubation, thewells were washed 3 times with PBS and the binders were eluted withacidic buffer (100 mM HCl, pH 2.2), and immediately neutralised with 2MTris solution. For the next panning round the eluted phage pools wereamplified by infecting E. coli XL-1 Blue cells. Five rounds of panningwere performed.

III. Characterisation of the Hevein-Binders

After the last panning cycle scFv phage display DNA was isolated andtransformed into E. coli HB2151 (supE⁻) cells in order to expresssoluble scFv fragments. Between the scFv sequence and the phage gene IIIsequence the scFv phage display vector contains TAG-amber stop codonwhich will be translated as glutamate in E. coli strains with supE⁺genotype but as a stop codon in E. coli strains with supE⁻ genotype.Sixty-two individual clones were grown in a small scale to producesoluble scFv fragments for preliminary characterisation. Clones wereanalysed on ELISA test using hevein-coated wells to catch thehevein-specific binders and control protein wells to see non-specificbinding (data not shown). Most of the clones bound With high affinity tohevein. Nineteen of the most promising clones were sequenced (Sanger etal., 1977) and two of them were selected for further characterisation(FIGS. 4 and 5).

EXAMPLE 2 Cloning and Characterisation of Human Fab Fragments withHevein-Binding Specificity

In this example the human IgE scFvs with hevein-binding specificity wereconverted to human Fab fragments with IgG1 subtype. Due to knowndifficulties in forming multimers, the 1A4 and 1C2 scFvs, obtained fromthe scFv antibody library, were cloned and bacterially expressed as Fabfragments (Holliger et al., 1993, Desplancq et al., 1994). The resultingantibody fragments were further characterised by a competitive ELISA.

I. Cloning of the Human Fab Fragments with Hevein-Binding Specificity

The Fd regions were amplified by overlapping PCR. The primers used forthe PCR are given in Table V.

The resulting cDNAs of the Fd region and light chains were cloned intothe bacterial expression vector, pKKtac and then transformed into E.coli RV308. Soluble Fab fragments designated to 1A4G and 1C2G wereproduced and the Fab fragments were purified by an introduced C-terminalhexahistidinyl tag on a Sepharose column with immobilised nickel to asubstantial purity (data not shown).

II. Characterisation of the Human Fab Fragments

The characterisation of the purified 1A4G and 1C2G was performed bycompetitive ELISA. First, increasing amounts of latex polypeptides,isolated from latex examination gloves according to Alenius andco-workers (1996), were incubated with the samples, 1A4G and 1C2G, andthen the reaction mixtures were applied onto microtitre plate wellscoated with allergenic GFP-hevein fusion protein. Preparation of latexpolypeptides have been analysed to contain high latex allergenicactivity (data not shown). FIG. 6 shows the result of the competitiveELISA. The binding of the 1A4G (FIG. 6 a) and 1C2G (FIG. 6 b) to heveincould be inhibited by adding increasing amounts of native hevein.

IgE antibodies bind specifically to allergenic epitopes. To study thebinding specificity of the 1A4G antibody in more detail a competitiveELISA with peptides comprising the allergenic epitopes was performed(FIG. 7). Banerjee and co-workers (1997) have studied the allergenicepitopes of hevein, and they found two potential allergenic epitopes,6-mer and 13-mer. In competitive ELISA the binding of the 1A4G to theimmobilised hevein was inhibited by using the peptides of the allergenicepitopes. These results obtained in different competitive ELISAsindicate that the antibodies isolated from the antibody library can bindspecifically to the recombinant hevein and the native hevein as well. Inaddition, the preliminary results demonstrate that the 1A4G antibodybinds specifically to the allergenic epitopes of hevein. TABLE I Primersused for cDNA synthesis and PCR amplifica- tion of the human IgE Fdregion. Cε1: 5′- GCTGAAGGTTTTGTTGTCGACCCAGTC -3′ Cε2: 5′-CACGGTGGGCGGGGTGAAGTCCC -3′ CεNotI: 5′-GAATGGTGCGGCCGCGCTGAAGGTTTTGTTGTCG -3′ VH1a: 5′-ATGGCCGCAGCTCAGGTKCAGCTGGTGCAG -3′ VH1b: 5′-ATGGCCGCAGCTCAGGTCCAGCTTGTGCAG -3′ VH1c: 5′-ATGGCCGCAGCTSAGGTCCAGCTGGTACAG -3′ Vh1d: 5′-ATGGCCGCAGCTCARATGCAGCTGGTGCAG -3′ VH2a: 5′-ATGGCCGCAGCTCAGATCACCTTGAAGGAG -3′ VH2b: 5′-ATGGCCGCAGCTCAGGTCACCTTGARGGAG -3′ VH3a: 5′-ATGGCCGCAGCTGARGTGCAGCTGGTGGAG -3′ VH3b: 5′-ATGGCCGCAGCTCAGGTGCAGCTGGTGGAG -3′ VH3c: 5′-ATGGCCGCAGCTGAGGTGCAGCTGTTGGAG -3′ VH4a: 5′-ATGGCCGCAGCTCAGSTGCAGCTGCAGGAG -3′ VH4b: 5′-ATGGCCGCAGCTCAGGTGCAGCTACAGCAG -3′ VH5a: 5′-ATGGCCGCAGCTGARGTGCAGCTGGTGCAG -3′ VH6a: 5′-ATGGCCGCAGCTCAGGTACAGCTGCAGCAG -3′ VH7a: 5′-ATGGCCGCAGCTCAGGTSCAGCTGGTGCAA -3′ VH1A: 5′-TTACTCGCGGCCCAGCCGGCCATGGCCGCAGCT -3′

TABLE II Primers used for cDNA synthesis and PCR amplifica- tion ofhuman kappa and lambda chains. Cκ1: 5′-AGGTAGGGCGCGCCTTAACACTCTCCCCTGTTGAAGC -3′ Vκ1a: 5′-ATGGCAGCGGCTRACATCCAGATGACCCAG -3′ Vκ1b: 5′-ATGGCAGCGGCTGMCATCCAGTTGACCCAG -3′ Vκ1c: 5′-ATGGCAGCGGCTGCCATCCRGATGACCCAG -3′ Vκ1d: 5′-ATGGCAGCGGCTGTCATCTGGATGACCCAG -3′ Vκ2a: 5′-ATGGCAGCGGCTGATATTGTGATGACCCAG -3′ Vκ2b: 5′-ATGGCAGCGGCTGATRTTGTGATGACTCAG -3′ Vκ3a: 5′-ATGGCAGCGGCTGAAATTGTGTTGACRCAG -3′ Vκ3b: 5′-ATGGCAGCGGCTGAAATAGTGATGACGCAG -3′ Vκ3c: 5′-ATGGCAGCGGCTGAAATTGTAATGACACAG -3′ Vκ4a: 5′-ATGGCAGCGGCTGACATCGTGATGACCCAG -3′ Vκ5a: 5′-ATGGCAGCGGCTGAAACGACACTCACGCAG -3′ Vκ6a: 5′-ATGGCAGCGGCTGAAATTGTGCTGACTCAG -3′ Vκ6b: 5′-ATGGCAGCGGCTGATGTTGTGATGACACAG -3′ Vκ/λ1: 5′-TTGTTATTGCTAGCTGCACAACCAGCAATGGCAGCGGCT -3′ Cλ1: 5′-AGGTAGGGCGCGCCTTATGAACATTCYGYAGGGGC -3′ Vλ1a: 5′-ATGGCAGCGGCTCAGTCTGTGCTGACTCAG -3′ Vλ1b: 5′-ATGGCAGCGGCTCAGTCTGTGYTGACGCAG -3′ Vλ1c: 5′-ATGGCAGCGGCTCAGTCTGTCGTGACGCAG -3′ Vλ2: 5′-ATGGCAGCGGCTCAGTCTGCCCTGACTCAG -3′ Vλ3a: 5′-ATGGCAGCGGCTTCCTATGWGCTGACTCAG -3′ Vλ3b: 5′-ATGGCAGCGGCTTCCTATGAGCTGACACAG -3′ Vλ3c: 5′-ATGGCAGCGGCTTCTTCTGAGCTGACTCAG -3′ Vλ3d: 5′-ATGGCAGCGGCTTCCTATGAGCTGATGCAG -3′ Vλ4: 5′-ATGGCAGCGGCTCAGCYTGTGCTGACTCAA -3′ Vλ5: 5′-ATGGCAGCGGCTCAGSCTGTGCTGACTCAG -3′ Vλ6: 5′-ATGGCAGCGGCTAATTTTATGCTGACTCAG -3′ Vλ7: 5′-ATGGCAGCGGCTCAGRCTGTGGTGACTCAG -3′ Vλ8: 5′-ATGGCAGCGGCTCAGACTGTGGTGACCCAG -3′ Vλ4/9: 5′-ATGGCAGCGGCTCWGCCTGTGCTGACTCAG -3′ Vλ10: 5′-ATGGCAGCGGCTCAGGCAGGGCTGACTCAG -3′

TABLE III Primers used for PCR amplification of the human variableregions of the heavy chain. VH1: 5′- ATTTACTCGAGTGAGGAGACGGTGACCAGGGTGCC-3′ VH2: 5′- ATTTACTCGAGTGAAGAGACGGTGACCATTGTCCC -3′ VH3: 5′-ATTTACTCGAGTGAGGAGACGGTGACCAGGGTTCC -3′ VH4: 5′-ATTTACTCGAGTGAGGAGACGGTGACCGTGGTCCC -3′ VH1A: 5′-TTACTCGCGGCCCAGCCGGCCATGGCCGCAGCT -3′

TABLE IV Primers used for PCR amplification of the human variableregions of the light chains. Vκ1: 5′- TTATAGAGCTCGACATCCAGATGACCCAGTCTCC-3′ Vκ2: 5′- TTATAGAGCTCGATGTTGTGATGACTCAGTCTCC -3′ Vκ3: 5′-TTATAGAGCTCGAAATTGTGTTGACGCAGTCTCC -3′ Vκ4: 5′-TTATAGAGCTCGACATCGTGATGACCCAGTCTCC -3′ Vκ5: 5′-TTATAGAGCTCGAAACGACACTCACGCAGTCTCC -3′ Vκ6: 5′-TTATAGAGCTCGAAATTGTGCTGACTCAGTCTCC -3′ Vκ7: 5′-TATAAGCGGCCGCACGTTTGATTTCCACCTTGGTCCC -3′ Vκ8: 5′-TATAAGCGGCCGCACGTTTGATCTCCAGCTTGGTCCC -3′ Vκ9: 5′-TATAAGCGGCCGCACGTTTGATATCCACTTTGGTCCC -3′ Vκ10: 5′-TATAAGCGGCCGCACGTTTGATCTCCACCTTGGTCCC -3′ Vκ11: 5′-TATAAGCGGCCGCACGTTTAATCTCCAGTCGTGTCCC -3′ Vλ1: 5′-ATTTAGAGCTCCAGTCTGTGTTGACGCAGCCGCC -3′ Vλ2: 5′-ATTTAGAGCTCCAGTCTGCCCTGACTCAGCCTGC -3′ Vλ3: 5′-ATTTAGAGCTCTCCTATGTGCTGACTCAGCCACC -3′ Vλ4: 5′-ATTTAGAGCTCTCTTCTGAGCTGACTCAGGACCC -3′ Vλ5: 5′-ATTTAGAGCTCCACGTTATACTGACTCAACCGCC -3′ Vλ6: 5′-ATTTAGAGCTCCAGGCTGTGCTCACTCAGCCGTC -3′ Vλ7: 5′-ATTTAGAGCTCAATTTTATGCTGACTCAGCCCCA -3′ Vλ8: 5′-ATATTGCGGCCGCACCTAGGACGGTGACCTTGGTCCC -3′ Vλ9: 5′-ATATTGCGGCCGCACCTAGGACGGTCAGCTTGGTCCC -3′ Vλ10: 5′-ATATTGCGGCCGCACCTAAAACGGTGAGCTGGGTCCC -3′

TABLE V Primers used for PCR amplification of the human Fd regions withIgE and IgG1 subtype. 5′Cε: 5′-GCTCACCGTCTCCTCAGCCTCCACACAGAGCCCATCCG-3′3′Cε: 5′- GCATTGCATTGCGGCCGCTTAATGGTGATGGTGATGATGGCTGAAGGTTTTGTTGTCGACCC-3′ 5′Cγ: 5′-GGTCACCGTCTCCTCAGCCTCCACCAAGGGCCC-3′ 3′Cγ:5′- TTTAGTTTATGCGGCCGCTTAATGGTGATGATGATGGTGACAAGATTTG GGCTCTGC-3′ 5′Vε:5′-TTACTCGCGGCCCAGCCGGCCATGGCCGCAGCT-3′ 3′Vε: 5′-TGAGGAGACGGTGACC-3′5′Cκ: 5′-GGGACACGACTGGAGATTAAAACTGTGGCTGCACCATCTGTC-3′ 3′Cκ:5′-AGGTAGGGCGCGCCTTAACACTCTCCCCTGTTGAAGC-3′ 5′Vκ:5′-ATGGCAGCGGCTGAAACGACACTCACGCAG-3′ and5′-TTGTTATTGCTAGCTGCACAACCAGCAATGGCAGCGGCT-3′ 3′Vκ:5′-TTTAATCTCCAGTCGTGTCCC-3′.References

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1. A monoclonal antibody belonging to an IgE subclass and having binding specificity to allergenic hevein, or a functional fragment or derivative thereof.
 2. The monoclonal antibody according to claim 1, wherein the fragment is a scFv fragment or a Fab fragment.
 3. The monoclonal antibody according to claim 2, wherein the scFv fragment is 1A4 or 1C2.
 4. An isolated DNA molecule encoding the monoclonal antibody or a fragment or derivative thereof according to any one of the preceding claims, and fragments of such DNA, which encode at least one antibody chain of said antibody or antibody derivative.
 5. The isolated DNA molecule according to claim 4, wherein the antibody chain is the Complementarity Determining Region (CDR) of the V_(L) and/or V_(H) region.
 6. The isolated DNA molecule according to claim 4 cloned into a vector.
 7. The isolated DNA molecule according to claim 6, wherein said vector is an expression vector capable of expressing antibodies, as well as fragments and derivatives thereof as claimed in any one of claims 1 to
 3. 8. A host cell containing a DNA according to claim
 4. 9. The host cell according to claim 8, capable of expressing a monoclonal antibody or a fragment or derivative thereof as claimed in any one of claims 1 to 3 or at least one antibody chain of said antibody or antibody derivative.
 10. The host cell according to claim 9, wherein the antibody chain is the scFv fragment as claimed in claim 2 or
 3. 11. A method of preparing a monoclonal antibody or a fragment or derivative thereof according to any one of claims 1 to 3, comprising the steps of culturing a host cell according to claim 8 capable of expressing at least one of the required antibody chains, and recovering said antibody or antibody fragment or derivative.
 12. The method according to claim 11, further comprising the steps of combining component chains after the recovery step, introducing combined component chains into a second host cell, and recovering said combined component chains.
 13. The method according to claim 11, further comprising the step of labelling said antibody or antibody derivative.
 14. A method of preparing a monoclonal antibody or a fragment or derivative thereof according to any one of claims 1 to 3, comprising the step of synthetically producing at least a portion of said antibody or antibody derivative.
 15. A phage or microbial cell, which presents an antibody fragment according to claim 2 as a fusion protein with a surface protein.
 16. A method of selecting an antibody fragment according to claim 2 or 3, comprising the steps selecting said antibody fragment from a display library of antibody fragments containing a phage or cell according to claim
 15. 17. A method of assaying hevein in a sample, comprising the steps of obtaining said sample, and assaying for hevein by employing a monoclonal antibody or a fragment or derivative thereof according to any one of claims 1 to
 3. 18. A test kit comprising an antibody or a fragment or derivative thereof according to any one of claims 1 to 3 in a suitable container for transport and storage.
 19. A monoclonal antibody or a fragment or derivative thereof according to any one of claims 1 to 3 for use in immunodiagnostics.
 20. A monoclonal antibody or a fragment or derivative thereof according to any one of claims 1 to 3 for use in immunotherapy. 